Characterization of a Vimentinhigh/Nestinhigh proteome and tissue regenerative secretome generated by human pancreas-derived mesenchymal stromal cells

Abstract

Multipotent/mesenchymal stromal cells (MSCs) exist within a variety of postnatal tissues; however, global proteomic analyses comparing tissue-specific MSC are limited. Using human bone marrow (BM)-derived MSCs as a gold standard, we used label-free mass spectrometry and functional assays to characterize the proteome, secretome, and corresponding function of human pancreas-derived MSCs (Panc-MSCs) with a classical phenotype (CD90+/CD73+/CD105+/CD45−/CD31−). Both MSC subtypes expressed mesenchymal markers vimentin, α-SMA, and STRO-1; however, expression of nestin was increased in Panc-MSCs. Accordingly, these Vimentin high /Nestin high cells were isolated from fresh human pancreatic islet and non-islet tissues. Next, we identified expression of >60 CD markers shared between Panc-MSCs and BM-MSCs, including validated expression of CD14. An additional 19 CD markers were differentially expressed, including reduced pericyte-marker CD146 expression on Panc-MSCs. Panc-MSCs also showed reduced expression of proteins involved in lipid and retinoid metabolism. Accordingly, Panc-MSCs showed restricted responses to adipogenic stimuli in vitro, although both MSC types demonstrated trilineage differentiation. In contrast, Panc-MSCs demonstrated accelerated growth kinetics and competency to proneurogenic stimuli in vitro. The secretome of Panc-MSCs was highly enriched for proteins associated with vascular development, wound healing and chemotaxis. Similar to BM-MSCs, Panc-MSCs conditioned media augmented endothelial cell survival, proliferation, and tubule formation in vitro. Importantly, the secretome of both MSC types was capable of stimulating chemotactic infiltration of murine endothelial cells in vivo and reduced hyperglycemia in STZ-treated mice following intrapancreatic injection. Overall, this study provides foundational knowledge to develop Panc-MSCs as a unique MSC subtype with functional properties beneficial in regenerative medicine for diabetes and vascular disease. K E Y W O R D S adipogenesis, multipotent stromal cells, nestin, pancreas, proteomics, regenerative medicine, secretome

Multipotent mesenchymal stromal cells derived from porcine exocrine pancreas improve insulin secretion from juvenile porcine islet cell clusters

“…Very recently, it had been shown that human pancreatic‐derived MSC are highly vimentin/nestin positive and produce a conditioned media highly enriched for proteins associated with vascular development, wound healing, and chemotaxis similar to human bone marrow‐derived MSC. Further, intrapancreatic injection of these MSC reduced hyperglycemia in streptozotocin‐treated mice 44 …”

Ultrafiltration Segregates Tissue Regenerative Stimuli Harboured Within and Independent of Extracellular Vesicles

“…Witwer et al recently highlighted the collective focus of ISCT and ISEV to further improve the guidelines for classifying MSC and MSC-generated EVs designed for therapeutic applications 26 , respectively. Indeed, the microenvironment of culture 61 and/or tissue source of origin [62][63][64][65] can lead to dynamic phenotypes and functional characteristics demonstrated by MSC in vitro 58,66 . It is also becoming evident that serial passaging and unwanted differentiation of MSC can lead to a loss of therapeutic activity 14,19,67 , such as the acquisition of a senescent secretome 68 .…”

“…All studies were approved by the Human Research Ethics Board at the University of Western Ontario (REB 12934, 12252E). Human pancreatic islets were provided by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) funded Integrated Islet Distribution Program (IIDP) at the City of Hope (California, USA), NIH Grant #2EC4DK098085-02.Panc-MSC were established, cultured and used to generate conditioned media (CM), as previously described 20 . CM was subsequently processed in one of three ways (Supplemental Figure 1).…”

“…As such, the capacity for MSC to stimulate tissue regeneration has been proposed as a defining characteristic to segregate MSC from nontherapeutic counterparts 18,19 . We recently conducted an in-depth proteomic characterization of MSC derived from human pancreatic tissue preperations (Panc-MSC) that shared classical MSC characteristics with BM-MSC, albeit distinct phenotypic, secretory and functional characteristics were observed in vitro 20 . Interestingly, both MSC-types generated a secretome that supported endothelial and pancreatic cell functions in vivo.…”

Porcine pancreas mesenchymal cell characterization and functional differentiation into insulin‑producing cells in vitro

“…Pancreas-derived mesenchymal stromal cells (PMSCs) are one of the types of mesenchymal cells isolated from pancreatic tissues. Compared with other cells types, PMSCs are highly enriched during vascular development, chemotaxis and wound healing ( 12 ). PMSCs also possess the ability to decrease hyperglycemia in type 1 diabetes model mice after intrapancreatic injection ( 12 ).…”